Acta Medica et BiologicaVol.55, No.l, 9-15, 2007

Glomerular Podocytes Express Protocadherin 17

Takuma TAKATA, Eishin YAOITA, Junichi KAMIIE, Huiping LI, Hidehiko FUJINAKA, Yutaka YOSHIDA, FumitakeGEJYO and Tadashi YAMAMOTO

D epartment of Structural Pathology, Institute of Nephrology, 2Division of Clinical Nephrology and Rheumatology, Graduate School of Medicaland Dental Sciences, Niigata University, Institute for Clinical Research, Niigata National Hospital, Niigata, Japan

Received December18, 2006; accepted December20, 2006

Summary. During a search for cadherin-relatedmolecules expressed in rat glomeruli by RT-PCR withdegenerate primers, wehave isolated CDNAencodingprotocadherin 17 (PcdhlT) in addition to Fatl.Although Pcdhl? has been suggested to participatein cell-cell adhesion and cell sorting, little is knownabout its distribution and function. To elucidate thelocalization and expression of Pcdhl?, ribonucleaseprotection assay and in situ hybridization wereperformed in the rat. Pcdhl7 expression was detecteddistinctly in RNAs from the cerebrum, cerebellum,lung, but spleen but weakly in RNAs from the wholekidney. In the kidney, intense signals for Pcdhl?werefound in glomerular RNAs, with only or weaksignals at best in cortical and medullary RNAs.In situhybridization showed that Pc*//f7 7 was predominantlyexpressed by podocytes in the glomerulus. Pcdhl?were found to have two isoforms that differ in thelength of the cytoplasmic domains. The expressionlevel of both isoforms did not change in puromycinaminonucleoside nephrosis where slit diaphragmsdisappear and newjunctional complexes are newlyformed. In conclusion, Pcdhl? is expressed bypodocytes, which may be involved in several types oftheir intercellular junctions.

Key words- cadherin; kidney; glomerular epithelialcell; podocyte; protocadherin 17.

INTRODUCTION

The outer aspect of the renal glomerurus consists ofvisceral epithelial cells - referred to as podocytes -exhibiting an elaborate morphology. Podocytes extendprimary processes from the cell body onto multiple

capillary loops and form a tight network of interdigitatingfoot processes on the glomerular basement membrane,allowing them to participate in the integration ofcapillaries into a glomerulus. In addition, the essentialglomerular structure is determined by podocytes, asshownin the metanephric culture where glomeruli arecomposedonly these cells.1}

Cell adhesion molecules are crucial for theestablishment and maintenance of tissue architecture.Cadherins are the major cell adhesion moleculesresponsible for Ca++-dependent cell-cell adhesion.Recent studies have identified a variety of cadherin-related proteins in different tissues of various organisms,and it is evident that these proteins constitute a largecadherin super family.2) This super family comprisesat least six sub families: classical or type-I cadherins,atypical or type-II cadherins, desmocollins, desmogleins,protocadherins, and Flamingo cadherins. In addition,several cadherins such as Fat and invertebrate cadherinsclearly occupy isolated positions in the cadherinsuperfami ly.

To examinecadherins expressed in adult rat glomeruli,wetried a homology polymerase chain reaction (PCR)using degenerate primers based on highly conservedextracellular domains of known cadherin molecules.Consequently, CDNAencoding Fatl was isolated fromthe PCR products: it has been shown that Fatl is afundamental component of intercellular junctions ofpodocytes including slit diaphragms.3'4) In the presentstudy, wehave isolated another CDNAclone encodingcadherin-related molecule, protocadherin 17 (Pcdhl 7\from the same PCRproducts. Protocadherins representthe largest sub family of the cadherin super family.5'6)Classical cadherins have five ectodomain repeats, atransmembranesegment, and a conserved cytoplasmicdomain that interacts with /?-catenin. In contrast,

Correspondence: Eishin Yaoita, Department of Structural Pathology, Institute ofNephrology, Graduate School of Medical and Dental Sciences, Niigata University,1-757 Asahimachi-dori, Niigata 951-8510, Japan.

9

Abbreviations- PAN, puromycin aminonucleoside; PCR, polymerase chainreaction; Pcdh, protocadherin.

10 T. Takata et al.:

protocadherins have six or more ectodomain repeats ― which are encoded by unusually large exons ― in addition to other sequence features that distinguish them from the classical cadherins, including distinct intracellular domains. The tissue distribution or function of Pcdh17 is still unknown. Our results clearly show that Pcdh17 is expressed exclusively by podocytes in the kidney.

MATERIALS AND METHODS

Animals

Adult Wistar rats, weighing in at 160 g - 220 g, and newborns were purchased from Charles River Japan (Atsugi, Kanagawa) and used in these experiments. The procedures for the present study were approved by the Animal Committee at Niigata University School of Medicine, and all animals were treated according to the Guidelines for Animal Experimentation of Niigata University.

Total RNA preparation

Glomeruli were isolated from the renal cortex fraction by the conventional sieving method as described previously.7) The isolated glomeruli, renal cortices, medullae, neonatal whole kidney, and other organs were homogenized in TRIzol (GIBCO BRL, Grand Island, NY, USA), and the total cellular RNA was extracted from these samples essentially according to manufacture's instructions.

Polymerase chain reaction

PCR was performed on the cDNA preparation from rat isolated glomeruli as a template using two degenerated oligonucleoside primers. These primers were designed on the highly conserved extracellular domain of known cadherins as reported by Sano et al..8) PCR products of approximately 130bp were size fractionated, cloned, and sequenced as previously described.3) Some of the clones contained cDNA showing a high homology with human PCDH17 (GenBank accession number BC028165). While referring to the human PCDH17 sequence and rat genome, 23 primer pairs were designed to amplify rat Pcdh17 cDNA, and RT-PCR and 5'/3' RACE (Roche Diagnostics GmbH, Mannheim, Germany) were repeated.

Ribonuclease protection assay

Ribonuclease protection assay was carried out as previously described.4) Antisense cRNA probes for mRNA of Pcdh17 and glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) were 32P-labeled by in vitro transcription, using the plasmids inserted with each cDNA: 244 bp corresponding to bp 2672-2429 of rat Pcdh17 coding sequence, and 114 bp corresponding to bp 674-787 of GAPDH (M17701) as a housekeeping gene. The detection and analysis of bands were performed by phosphor imaging techniques using Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA, USA). The results of ribonuclease protection assay are represented as the ratio of Pcdh17 to GAPDH.

In situ hybridization

In situ hybridization was essentially carried out using a Ribomap kit and Discovery Automatic Staining Module (Ventana Medical Systems, Tucson, AZ), generally according to manufacture's instructions. Briefly, rat kidneys were fixed in 4% paraformaldehyde in PBS for three days at 4 ºC. Serial 6-μm kidney sections were automatically deparaffinized, fixed, and acid treated. Then the tissue sections were subjected to cell conditioning and protease digestion. Digoxigenin-labeled antisense and sense cRNA probes were synthesized by in vitro transcription using the linearized templates (422 bp corresponding to bp 1248-1669 of the rat Pcdh17 coding sequence). Hybridization was performed with an antisense or sense probe (30 ng/side) at 60 ºC for six h in Ribohybe hybridization solution (Ventana Medical Systems). Hybrids were detected with a alkaline phosphatase-conjugated antidigoxigenin antibody (Roche Diagnostics, Penzberg, Germany). Signal detection was performed with nitro blue tetrazolium chloride 5-bromo-4-cloro-3-indolyl phosphate 4-toluidinium salt as a chromogenic substrate at room temperature for three h in the dark.

Induction of experimental nephrosis in Wistar rats

Puromycin aminonucleoside (PAN) nephrosis was induced in four rats by an intravenous injection of PAN (Sigma) at a dose of 5 mg/100 g body weight in phosphate-buffered saline. Nephrotic rats were sacrificed 10 days after the PAN injection. Glomeruli isolated from the four rats were pooled and used as one sample of glomerular RNA. Measurements of 24 h urinary protein levels in PAN nephrosis revealed that a single intravenous injection of PAN induced massive proteinuria in WKY rats five (33.6 + / -22.7 mg/day) and 10 days (137.8+ / -49.6 mg/day) after the injection. Urinary protein excretion before PAN injection was 2.8+ / -0.9 mg/day.

11Protocadherin 17 in Podocytes

RESULTS

Organ distribution of Pcdh17 mRNA

To clone cadherins expressed in rat glomeruli, PCR was performed on glomerular cDNA. PCR products of a predicted size of approximately 130bp were puriied and cloned. Sequencing the clones and homology research revealed some of the clones to exhibit a sequence highly similar to human PCDH17. To confirm the expression of Pcdh17 in rat glomeruli and the other organs, a cRNA probe for rat Pcdh17 was prepared, and ribonuclease protection assay was performed (Fig. 1). Signals for Pcdh17 mRNAs were detected distinctly in RNAs from the cerebrum, cerebellum, lung, and spleen, but weakly in RNAs from the whole kidney. When kidneys were fractionized into glomeruli, cortices, and medullae, intense signals were found in glomerular RNAs with weak signals in cortices and medullae, indicating that Pcdh17 expression was restricted to glomeruli in the kidney.

Localization of Pcdh17 expressing cells in the

glomerulus

To elucidate which cells in the glomerulus express Pcdh17, the distribution of Pcdh17 mRNA was accessed by in situ hybridization (Fig. 2). Pcdh17 mRNA was observed predominantly in glomerular cells facing the Bowman's space; namely, the podocytes.

Deduced amino acid sequence of rat Pcdh17

RT-PCR and 5'/3' RACE using cDNA from rat glomeruli were carried out referring to the human PCDH17 sequence and rat genome. We have finally confirmed a 3875 bp cDNA sequence which covered the regions from the full length of the coding sequence to a poly A tail. Pcdh17 was found to be 1159aa in length, with the predicted molecular mass of 126 kDa (Fig. 3). The amino acid sequences exhibited an extensive homology to the human PCDH17, possessing 98% identity. The predicted amino acid sequences began with a 17aa hydrophobic stretch, suggesting this to be the signal peptide of the

Fig. 1. Expression of protocadherin 17 mRNA detected by ribonuclease protection assay using 10 μg of total RNA isolated from several rat organs. Ten μg of yeast tRNA was used as a negative control.

12 T. Takata et al.:

Fig. 3. Deduced amino acid sequence of rat protocadherin 17. The boxes (CM-1 and CM-2) are comparatively common intracellular motif in δ-protocadherins. The grey-colored region in the cytoplasmic domain is the part absent in the alternative splice variant. EC, ectodomain; TM, transmembrane domain; CP, cytoplasmic domain.

Fig. 2. Localization of protocadherin 17 mRNA in the rat glomerulus detected by in situ hybridization using a digoxigenin-labeled cRNA probe. Podocytes (arrows) show a distinct expression of protocadherin 17 when hybridized with the antisense probe A. but not with the sense probe B.

13Protocadherin 17 in Podocytes

Fig. 5. Expression of protocadherin 17 mRNA detected by ribonuclease protection assay using 10 μg of total RNA from normal and PAN nephrotic glomeruli and 20 μg of total RNA from adult and neonatal whole kidneys. Twenty μg of yeast tRNA was used as negative control. The cRNA probe contained the region corresponding to the second exon.

Fig. 4. Genomic organization of rat protocadherin 17 gene, its transcripts, and the encoded proteins. Protocadherin 17 has at least two isoforms due to alternative splicing of the second exon. The deletion of the second exon produces a stop codon (TAG), resulting in the short cytoplasmic domain.

14 T. Takata et al.:

protein. The extracellular domain at the N-terminal side consisted of six cadherin repeats. A twenty-one hydrophobic stretch corresponded to the transmembrane domain. The cytoplasmic domain contained CM-1 and CM-2 motifs that are a comparatively common intracellular motif in protocadherins.9)

We found an alternative splice variant when PCR was done in the cytoplasmic region. The valiant was lacking in the region corresponding to the second exon (59 b) (Fig.4). The deletion caused a stop codon to appear just after the sequence corresponding to the irst exon, generating the isoform with a short cytoplasmic domain which lacked CM1 and CM2 (Fig.3). The presence of the isoforms was supported by the protein expression experiment using the above PCR products ligated to the expression vector (pGEX-2T). The molecular mass difference of the fusion proteins from the two PCR products was approximately 34 kDa in the electrophoresis, which is coincident with that of the predicted molecular mass between the isoforms (data not shown).

Pcdh17 expression in the nephrosis and the neonatal

kidney

A cRNA probe including the second exon was prepared (Fig.4), and a ribonuclease protection assay was performed to examine whether the expression levels of the isoforms change in nephrosis or development (Fig. 5). The Pcdh17 transcript in PAN nephrotic glomeruli were on the same level as that in normal glomeruli. The neonatal kidney showed a twice higher level of expression than the adult kidney. The expression level of the isoform with the second exon was four or ive times higher than that of the isoform without exon 2 in every sample.

DISCUSSION

We have identified Pcdh17 in a screen for cadherins expressed in the rat glomerulus. Pcdh17 is a poorly characterized member of the protocadherin family. This is the first report concerning its organ distribution and the presence of isoforms. Pcdh17 expression was signiicantly detected in the central nervous system, lung, spleen, and glomerulus. One of the isoforms was devoid of a large part of the cytoplasmic domain. Pcdh17 belongs to the δ2-protocadherins that are a protocadherin subgroup recently identified.10,11)

Protocadherins are divided into clustered and non-clustered protocadherins.12) The former (α-, β- and γ-protocadherins) are encoded by genes which are clustered in a small genomic region. The latter genes are

scattered in the genome solitarily or as a small group encoding a few protocadherins. Nine out of the 14 non-clustered protocadherins share conserved cytoplasmic motifs (CM1 and CM2), which were first identified by homologies between Pcdh8, Pcdh10, Pcdh18, and Pcdh19.6,9) These proteins constitute the newly defined subfamily of δ-protocadherins that can be further subdivided into δ1- and δ2-protocadherins based on the presence (δ1) or absence (δ2) of another cytoplasmic conserved motif (CM3).10,11) The CM3 motif was shown to specially interact with protein phosphatase-1α. The structural features of Pcdh17 coincide with δ2-protocadherins. In contrast to the clustered protocadherins ― which are mostly expressed in the central nervous system, δ-protocadherins are additionally expressed in a variety of tissues such as the primitive streak, paraxial mesoderm, kidney, heart, and lung.9,10) Although several protocadherins have been reported to be expressed in the kidney by Northern blot analysis, Pcdh17 is unique in the respect that its expression is restricted to the glomerulus. In addition to the standard molecular structure, Pcdh17 had another isoform whose cytoplasmic domain was short, due to the deletion of the second exon and the resultant stop codon. The isoform contained neither CM1 nor CM2. δ-Protocadherins frequently show alternative RNA splicing leading to a truncated cytoplasmic domain. Hardly anything is known about the functional implications of the different isoforms. However, differential expression patterns of alternative splice variants of δ-protocadherins have been reported for Pcdh8 and PCDH11.13,14) In addition, it has been demonstrated in Xenopus that cell sorting activity decreases, when axial protocadherin (a Pcdh1 ortholog) or paraxial protocadherin (a Pcdh10 ortholog) has the cytoplasmic region intact, suggesting a negative regulation by the cytoplasmic region.15,16) The identiication of more molecules that link the cytoplasmic domains to signaling pathways and to the cytoskeleton will be necessary to clarify the functional signiicance of the isoforms. In situ hybridization demonstrated that Pcdh17 was expressed exclusively by podocytes. δ-Protocadherins resemble classic cadherins in their ability to mediate cell adhesion by preferring homophilic over heterophilic interactions, although binding appears to generally weak.11) Some of them have been shown to localize to cell-cell junctions or synapses. Based on the general features of the protocadherins, it is proper to assume that Pcdh17 localizes to cell-cell contact sites or intercellular junctions of podocytes. Slit diaphragms are the main intercellular junctions of podocytes under physiological conditions. Under the nephrotic condition, slit diaphragms are dislocated or disappear, while junctional complexes containing tight junctions and gap junctions are newly formed between foot processes.17)

15Protocadherin 17 in Podocytes

The ribonuclease protection assay revealed that the Pcdh17 expression level did not significantly change in PAN nephrosis. This unchanged expression in PAN nephrosis is similar to that of Fat1, another member of the cadherin superfamily expressed by podocytes.4) Fat1 is a component shared by slit diaphragms and the newly formed junctional complexes in the nephrosis. In addition, Fat1 is involved in the intercellular junctions of immature podocytes. The Fat1 transcript level in the neonatal kidney is significantly higher than that in the adult kidney, which is also similar to Pcdh17 expression in the neonatal kidney. Thus, we presume that Pcdh17

exhibit the same distribution in podocytes as Fat1. We tried to prepare antibodies against Pcdh17 using three oligopeptides corresponding to three different cytoplasmic regions and two GST fusion proteins of the extracellular and cytoplasmic domains. Unfortunately, every antibody showed low titers and several bands in immunoblots even after the affinity purification. In the immunohistochemistry using these antibodies, we could not find any significant staining localized in the glomerulus. The precise localization and function of Pcdh17 in podocytes will be elucidated when the speciic antibody established. In conlusion, Pcdh17 is expressed predominantly by podocytes in the kidney and may be involved in several types of intercellular junctions of podocytes.

Acknowledgments. The work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Ministry for Education, Science, and Culture (17590822).

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